Acute myeloid leukemia (AML) is an aggressive disease that is characterized by an abnormal level of immature myeloblasts in the blood and bone marrow. FLT3 is a receptor tyrosine kinase that plays an integral role in haematopoiesis, and alteration to this cohesive signaling machinery leads to haematopoietic malignancies including AML. In fact, FLT3 is implicated as a major factor in AML relapse (Leick, M. B. & Levis, M. J. Curr. Hematol. Malig. Rep. 1-15 (2017). doi:10.1007/s11899-017-0381-2).
Thirty percent of AML cases have been shown to have mutations to FLT3 causing a conformational change that leads the kinase to be constitutively active (Stirewalt, D. L. & Radich, J. P. Nat. Rev. Cancer 3, 650-65 (2003); and Pozarowski, P. & Darzynkiewicz, Z. Methods Mol. Biol. 281, 301-11 (2004)). Mutations to the juxtamembrane and the kinase domain are the most common types of mutations (Stirewalt, D. L. & Radich, J. P. Nat. Rev. Cancer 3, 650-65 (2003); Leung, A., et al., Nat. Rev. Leuk. 27, 260-268 (2013); and Yamamoto, Y. Blood 97, 2434-2439 (2001)). Internal tandem duplication (ITD) to the juxtamembrane domain or the first TKD occurs when a segment is duplicated (head to tail) leading to the loss of repressive regions of the RTK. Unlike WT FLT3, FLT3-ITD has also been implicated in the up-regulation of the pro-survival STATSA signaling pathway (Yoshimoto, G. et al. Blood 114, 5034-5044 (2009)). A second common mutation is a point mutation of aspartic acid 835 to a tyrosine residue in the kinase domain (TKD), which also leads to a constitutively active kinase. The ITD and TKD mutants also can activate and dimerize with the wild type (WT) FLT3. The effects of these mutations on FLT3 signaling are still unclear, but one possibility is that mutant FLT3-TKD and FLT3-ITD activate alternative signaling pathways, or activate standard FLT3 pathways aberrantly, compared to the WT.
Early studies have shown that mutations to FLT3 are correlated with poor long-term prognosis (Swords, R., Freeman, C. & Giles, F. Leukemia 26, 2176-2185 (2012); Kim, Y. et al. Nature 5, e336-7 (2015); Hospital, M.-A. et al. Onco. Targets. Ther. 10, 607-615 (2017); and Lagunas-Rangel, F. A. & Chavez-Valencia, V. Oncol. 34, 114 (2017)). Patients with mutations to FLT3 initially achieve similar disease remission to those with the endogenous FLT3 but have an increased risk for relapse (Stirewalt, D. L. & Radich, J. P. Nat. Rev. Cancer 3, 650-65 (2003); Swords, R., Freeman, C. & Giles, F. Leukemia 26, 2176-2185 (2012); Lagunas-Rangel, F. A. & Chavez-Valencia, V. Oncol. 34, 114 (2017); and Smith, C. C., et al., Leukemia 1-3 (2015). doi:10.1038/1eu.2015.165). In vitro studies have shown that FLT3-ITD mutant expressing cell lines induce resistance to cytosine arabinoside, (Ara-C) which is a primary AML, therapeutic (Swords, R., Freeman, C. & Giles, F. Leukemia 26, 2176-2185 (2012)). These findings prompted the use of a combinatorial approach to AML therapies that included both Ara-C and FLT3 inhibitors but, unfortunately, use of FLT3 inhibitors leads to relapse by inducing new TKD mutations (Swords, R., Freeman, C. & Giles, F. Leukemia 26, 2176-2185 (2012); and Hospital, M.-A. et al. Onco. Targets. Ther. 10, 607-615 (2017).
Computational modeling suggests that internal tandem duplication of the juxtamembrane domain or point mutation to aspartic acid 835 (D835Y) both alter the protein structure, leading to decreased potency for some FLT3 inhibitors (Smith, C. C., et al., Leukemia 1-3 (2015). doi:10.1038/1eu.2015.165). Based on this clinical importance, discovery of alternative inhibitors and clinical monitoring of inhibitor efficacy are of interest to physicians treating AML patients with FLT3 overexpression and/or mutations. Assays that could be used in these capacities would therefore be valuable, however existing peptide substrates are either not specific (e.g. the “Abltide” substrate) or not efficiently phosphorylated (e.g. the substrate reported by Böhmer, F.-D. & Uecker, A. Br. J. Haematol. 144, 127-30 (2009), which was poorly phosphorylated in our experience). More optimal peptide substrates would benefit drug discovery and assay development efforts for FLT3 and its variants.
Currently there is a need for agents that are substrates for specific kinases (e.g. FLT3 kinase).